N-terminal sequence analysis of polypeptide at the picomole level.

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.     

Amino acid analysis by dinitrophenylation and reverse-phase high-pressure liquid chromatography

The determination of amino acids has been achieved by reverse-phase high-pressure liquid chromatography of their dinitrophenyl derivatives. The methods developed permit the quantitation of all amino acids commonly encountered in a protein hydrolysate and the effect of various parameters on this separation was systematically evaluated. The procedure eliminates the need for specialized postcolumn equipment as employed in conventional amino acid analysis and can be obtained by a simple gradient high-pressure chromatograph. The sensitivity obtained is comparable to that available by methods in common usage, being able to determine amino acids quantitatively in the low picomole range.     

Optimized isocratic conditions for the simultaneous determination of serotonin precursors and metabolites by reversed-phase high-pressure liquid chromatography with electrochemical detection

We describe an analytical procedure for the simultaneous determination of 5-hydroxyindole derivatives using high-pressure liquid chromatography with electrochemical detection. The procedure clearly resolves 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxytryptophol, and 5-hydroxyindole-3-acetic acid. The C-18 extraction column methodology and high-pressure liquid chromatography-electrochemical detection parameters have been developed to provide a rapid, sensitive, and reproducible quantitative determination of these 5-hydroxyindoles with picogram sensitivity. Chromatograms obtained from the analysis of whole normal mouse brain by the present technique clearly resolve the 5-hydroxyindoles and appear to be uncomplicated by interfering substances.     

High-pressure liquid chromatographic identification of phenylthiohydantoin derivatives of all twenty common amino acids

Phenylthiohydantoin (PTH) derivatives of all 20 common amino acids can be separated by high-pressure liquid chromatography. By using a Waters reversed-phase C₁₈ column eluted with a concave ethanol gradient in ammonium acetate, pH 5.1, all PTH derivatives were eluted in less than 30 min. The NH₂-terminal amino acid sequence of the human retinolbinding protein could unambiguosly be established for the first 40 residues. Likewise, HLA-DR antigens biosynthetically labeled with [³H]tyrosine and [³H]phenylalanine were subjected to automatic sequential degradation. Labeled PTH-amino acids were easily identified by the described chromatographic procedure.     

Analysis of demethylaminoazobenzene-thiohydantoins of amino acid by high-pressure liquid chromatography

Dimethylaminoazobenzene-thiohydantoins of amino acid can be quantitatively analyzed by high-pressure liquid chromatography at picomole level. As little as 5 to 10 pmol of dimethylaminoazobenzene-thiohydantoins of amino acid can easily be detected in the visible region (436 nm) against a stable baseline. Three amino acid pairs, namely glutamine and threonine, methionine and proline, and leucine and isoleucine, have not yet been separated. This new technique provides a sensitive and efficient tool for measuring the recovery of amino terminal amino acids using the dimethylaminoazobenzene-isothiocyanate method and the repetitive yield of sequence determination using the dimethylaminoazobenzene-isothiocyanate phenylisothiocyanate double-coupling method.     

Analysis of derivatives of carbohydrates by high-pressure liquid chromatography

The behaviour of benzoylated derivatives of alditols, monosaccharides, disaccharides, amino sugars, and methyl glycosides in high-pressure liquid chromatography (h.p.l.c.) has been investigated. A system was devised, using the most basic equipment of a single pump and fixed-wavelength u.v. detector, which gave good separations of the components of mixtures of derivatised methyl glycosides. Fractionation of complex mixtures of many of the other benzoylated carbohydrates was achieved in less than 30 min. The 4-nitrobenzoates were less useful for routine analyses.     

Separation of amino acid phenylthiohydantoin derivatives by high-pressure liquid chromatography

Separation of the phenylthiohydantoin (PTH) derivatives of all 20 common amino acids is accomplished in approximately 11 min with excellent resolution by using high-pressure liquid chromatography. The chromatography is achieved at 50 degrees C on an Altex reversed-phase PTH-C18 column in an ammonium acetate-buffered acetonitrile, pH 4.5, mobile phase. Simple isocratic and linear gradient steps are used. Retention times for the various PTH-amino acids are very reproducible. Because the baseline is flat and free of background noise, PTH-amino acids can be detected in the low picomole range. The simplicity of this chromatographic system allows it to be easily automated.     

Analyses of phenolic and flavonoid compounds by high-pressure liquid chromatography

A technique to separate complex phenolics extracted from plant tissue has been developed using high-pressure liquid chromatography. The compounds, in glycosidic and ester linkages, can be collected as separation occurs. After hydrolysis, flavonoid components as well as the benzoic and cinnamic acid derivatives can be separated and identified with one chromatographic analysis of tissue samples. These techniques may be helpful in toxicity studies and in determining the role of phenolics in plants.     

Rapid determination of doxorubicin and its fluorescent metabolites by high pressure liquid chromatography.

An original method for the separation and quantitation of doxorubicin (DOX) and its metabolites by high-pressure liquid chromatography and fluorometry is described. Doxorubicin and its derivatives are extracted from biological samples in a rapid, non-destructive manner, with a recovery close to 100%. The different compounds are rapidly separated by high-pressure liquid chromatography using an eluant system containing magnesium chloride, and detected quantitatively by fluorometry down to a concentration of 1.5 ng/ml in less than 5 min. Using this method, we have determined doxorubicin and its metabolites in plasma and urine, after an intravenous injection into DBA₂ and NMRI mice.     

High-pressure liquid chromatographic analysis of amino acid phenylthiohydantoins: Comparison with other techniques

High-pressure liquid chromatography, utilizing reverse phase μ Bondapak C₁₈ columns and elution with increasing acetonitrile concentrations, has been used to resolve amino acid phenylthiohydantoins obtained from the automated Edman degradation of proteins. Assignment of identity to residues which are difficult to distinguish or identify conclusively by other conventional techniques is easily achieved by high-pressure liquid chromatographic techniques. The use of high-pressure liquid chromatography, in parallel with gas-liquid and polyamide thin-layer chromatography, allows unequivocal assignments of identity to amino acid phenylthiohydantoins obtained in protein sequencing. Single protein sequence determinations can be extended by 20 to 100% by the use of high-pressure liquid chromatography with rapid, accurate, and quantitative identifications of amino acid phenylthiohydantoins.     

A modified system for thiazolinone conversion to thiohydantoin derivatives and their separation by high-pressure liquid chromatography

An improved and very simple procedure for thiazolinone conversion to thiohydantoin derivatives and their separation by reverse-phase high-pressure liquid chromatography is described. Trifluoroacetic acid (10%) in ethyl acetate has been employed as a conversion reagent to circumvent the deamidation of acid amides and methylation of acidic amino acids, with a concomitant increase in the detection limits of these residues. Additionally, a very simple procedure has been developed for the separation of phenylthiohydantoin (PTH) derivatives of amino acids. The system takes advantage of the computer-controlled precise mixing of the solvents A and B to achieve accurate pH and thus avoid the necessity of pH adjustment of a buffer. The procedure is simple and highly reproducible, and separates all the 20 known PTH amino acids. The efficiency of the method has been examined on synthetic and natural proteins/peptides, in manual and autoconversion systems, over a period of more than 18 months.     

Reverse-phase liquid chromatographic analysis of amino acids after reaction with o-phthalaldehyde

Precolumn formation of o-phthalaldehyde (OPA) derivatives of amino acids, followed by reverse-phase separation and fluorescent detection, provides rapid, sensitive amino acid analysis. Eighteen OPA-amino acid derivatives are resolved on a Micropack MCH 5 column and can be measured at picomole levels. Ease of derivative preparation and separation makes liquid chromatographic analysis of OPA-amino acids a convenient and improved technique for measuring or confirming the presence of low levels of amino acids in aqueous solutions. Use of the method was demonstrated by measuring low concentrations of amino acids released from zooplankters stressed by contaminants.     

Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography

The conditions for tryptic digestion and subsequent peptide mapping of the ATP-dependent proteolysis cofactor ubiquitin and its derivatives are described. In aqueous solution, the native ubiquitin which is composed of 76 amino acids undergoes only a single cleavage at arginine-74. Full digestion of ubiquitin was obtained in 6.5 M urea, although cleavages at lysine-33 and arginine-74 were slow. Peptide mapping was achieved by reverse-phase high-performance liquid chromatography with a C18 column using a trifluoroacetic acid/triethylamine buffer system and acetonitrile as eluants. The peptides, separated using a linear gradient, were identified by amino acid analysis. Derivatives analyzed by this method include oxidized, monoiodotyrosyl, and diiodotyrosyl ubiquitin. This technique will be useful in examining peptides of chemically modified ubiquitin with respect to extent and specificity of modification. In addition, this technique will be useful in comparing ubiquitin peptides of different organisms.     

Analysis of sugars in glycoproteins by high-pressure liquid chromatography

A method for analyzing the carbohydrate composition of glycoproteins and similar glycoconjugates by methanolysis followed by reverse-phase high-pressure liquid chromatography of the perbenzoylated methyl glycosides has been developed. As described, the method is capable of quantifying sugars in the 1- to 10-nmol range while further optimization of procedures may increase the usable sensitivity by a factor of 10 or greater. Improved yields of the sugar derivatives have been achieved by incorporating several modifications of the original methanolysis procedure. This, together with the use of high-pressure liquid chromatography rather than gas chromatography for separating the sugar derivatives, eliminates the need for empirically determined molar response ratios.     

Measurement of branched chain amino acids in blood plasma by high performance liquid chromatography

A simple and rapid high performance liquid chromatographic technique is described for the separation and quantitation of plasma branched chain amino acids. After addition of a norleucine internal standard, plasma samples are acidified with acetic acid, and amino acids are separated from proteins and other plasma components by passage of the acidified plasma through an ion exchange resin. The ammonium hydroxide eluate from the resin is dried, phenylisothiocyanate derivatives are prepared, and the amino acids are separated on a Waters reverse-phase "Pico-Tag" column with an ultraviolet detector set at 254 nm. In addition to the branched chain amino acids (leucine, valine, and isoleucine), aspartate, glutamate, serine, threonine, alanine, and methionine are quantitated with high precision and accuracy, as verified by quantitative recovery and comparison with an automatic amino acid analyzer. The advantages of the method are its simplicity, speed, stability of derivatives, high reproducibility, low per-sample cost, and the use of a simple fixed-wavelength ultraviolet detector.     

Analysis of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acids at sub-picomole levels by high-performance liquid chromatography: simultaneous manual sequencing of picomole quantities of several polypeptides

A single-column high-performance liquid chromatographic separation of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives, generated during polypeptide sequence analysis by the 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate/phenylisothiocyanate double coupling technique, is described. Recovery of the serine and threonine derivatives was improved by substituting boron trifluoride-diethyl etherate for trifluoroacetic acid in the thiazolinone cleavage reactions. Residues, including the S-carboxymethyl derivative of cysteine, were assigned after a single injection and a cycle time of 30 min. Quantities of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives as low as 100 fmol were detected. Interference of sequencing artefacts with residue assignment was avoided. This technique allows simultaneous manual sequencing of several proteins or peptides at the level of a few picomoles.     

Chemical synthesis of biologically active tat trans-activating protein of human immunodeficiency virus type 1.

Full-length (86-residue) polypeptide corresponding to the human immunodeficiency virus type 1 tat trans-activating protein was chemically synthesized on a semiautomated apparatus, using an Fmoc amino acid continuous-flow strategy. The bulk material was relatively homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, and it showed trans-activating activity when scrape loaded into cells containing a human immunodeficiency virus long terminal repeat-chloramphenicol acetyl-transferase reporter plasmid. Reverse-phase high-pressure liquid chromatography yielded a rather broad elution profile, and assays across the column for biological activity indicated a sharper peak. Thus, high-pressure liquid chromatography provided for enrichment of biological activity. Fast atom bombardment-mass spectrometry of tryptic digests of synthetic tat identified several of the predicted tryptic peptides, consistent with accurate chemical synthesis.     

Analysis by reverse-phase high-pressure liquid chromatography of phenylisothiocyanate-derivatized 1-aminocyclopropane-1-carboxylic acid in apple extracts

A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues is described. This method is based on the derivatization of ACC with phenylisothiocyanate, and the subsequent separation and quantification of the resulting phenylthiocarbamyl-ACC by reverse-phase high-pressure liquid chromatography. Phenylthiocarbamylation of ACC (and other amino acids) in apple extracts is complete within 20 min at room temperature. After removing solvents and reagent, the phenylthiocarbamyl derivatives are separated on an octadecyl reverse-phase column, eluted with a mixture of acetonitrile and sodium acetate buffer at pH 4.6, and monitored with a uv detector set at 254 nm. An analysis of apple extract can thus be achieved in 23 min and detect quantities as low as 1 pmol. Assays have been done to compare the efficiency of this method with that of a method using an ion-exchange amino acid analyzer and with that of Lizada and Yang's method [(1979), Anal. Biochem. 100, 140-145]. The latter method proved to yield markedly less accurate results than the other two, but the derivatization-HPLC method was preferred because of simplicity of operation and a better separation of ACC.     

Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.     

High-performance liquid chromatographic analysis of physiological amino acids in human brain tumors by pre-column derivatization with phenylisothiocyanate

A reversed-phase high-performance liquid chromatographic technique for the determination of free amino acids in five biopsies of human brain tumors (two meningiomas, one glioblastoma and two oligodendrogliomas) is described. The frozen tissues were homogenized, deproteinized with perchloric acid and neutralized with potassium hydroxide. Aliquots of the supernatant containing the physiological amino acids are used for pre-column derivatization with phenylisothiocyanate. The derivatized PTC-amino acids (phenylthiocarbamyl derivatives) are stable for a five day period if stored as a powder at −20°C in an inert atmosphere and they can be analyzed on a reversed-phase column (PicoTag) using a gradient of two eluents with absorption detection at a wavelength of 254 nm. Good resolution of several amino acids (>30) is achieved within ca. 60 min. For most amino acids this method is suitable for an accurate measurement over a wide range of physiological concentrations (50–400 pmol) starting from a very small amount of sample.